Most organic reactions are carried out between molecules in the ground state. However, photochemical reactions, utilizing light of a specific wave-length range, promote molecules to an electronically excited state. Electrons can move from the ground-state energy level of the molecule to a higher level with this application of outside energy..sup.1 The following table (March, supra, page 210) illustrates physical processes undergone by excited molecules:.sup.2
______________________________________ S.sub.0 + h.nu. .fwdarw. S.sub.1.sup..nu. Excitation S.sub.1.sup..nu. Vibrational relaxation S.sub.1 .fwdarw. S.sub.0 + h.nu. Fluorescence S.sub.1 S.sub.0 + heat Internal conversion S.sub.1 T.sub.1.sup..nu. Intersystem crossing T.sub.1.sup..nu. Vibrational relaxation T.sub.1 .fwdarw. S.sub.0 + h.nu. Phosphorescence T.sub.1 S.sub.0 + heat Intersystem crossing S.sub.1 + A.sub.(S0) .fwdarw. S.sub.0 + A.sub.(S1) Singlet-singlet transfer (photosensitization) T.sub.1 + A.sub.(S0) .fwdarw. S.sub.0 + A.sub.(T1) Triplet-singlet transfer (photosensitization) ______________________________________
Some compounds will assume excited triplet.sup.3 states upon "excitation" by exposure to a certain wave-length of light. These compounds ("sensitizers" or "photosensitizers") can interact with various other compounds ("acceptors") and transfer energy to or electrons from the acceptors, thus returning the sensitizer to its unexcited or Found state. Most compounds will assume the excited singlet.sup.4 state upon "excitation." A photosensitizer in its triplet state is capable of converting ground-state oxygen (a triplet) to an excited singlet state. The singlet oxygen can result in the production of a detectable "signal" which can be monitored and/or quantitated. In the context of this invention, a sensitizer is a molecule with a Chromophore capable of absorbing light so that it becomes electronically excited. The best sensitizers are those which undergo Intersystem Crossing to the triplet state, i.e., involves the sequence: S.sub.0 +h.nu..fwdarw.S.sub.1.sup..nu., S.sub.1 T.sub.1.sup..nu., and T.sub.1 +A.sub.(S.sbsb.0.sub.) .fwdarw.S.sub.0 +A.sub.(T.sbsb.1.sub.). FNT .sup.1 According to March, Advanced Organic Chemistry, 3rd Ed., 1985, John Wiley & Sons, New York, N.Y., at p. 202: "Since the energy levels of a molecule are quantized, the amount of energy required to raise an electron in a given molecule from one level to a higher one is a fixed quantity. Only light with the frequency corrosponding to this amount of energy will cause the electron to move to the higher level. If light or another frequency (too high of too low) is sent through a sample, it will pass out without a loss in intensity, since the molecules will not absorb it. However, if light of the correct frequency is passed in, the energy will be used by the molecules for electron promotion and hence the light that leaves the sample will be diminished in intensity or altoghter gone." At page 204, March points that though promotion of an electron to either a singlet or triplet excited state would seemingly be possible "depending upon the amount of energy added" this is often not the case because certain transition are "forbidden." For example, singlet-triplet and triplet-singlet transitions are forbidden, "whereas singlet-single and triplet-triplet transitions are allowed." FNT .sup.2 The superscript .nu. indicated vibrationally excited state: excited state higher than S.sub.1 or T.sub.1 are omitted. FNT .sup.3 The condition within a molecule in which two unpaired electrons have the same spin. FNT .sup.4 The condition within a molecule in which all spins are paired.
The development of singlet oxygen is reviewed in "Singlet Molecular Oxygen," edited by A. Paul Schaap (Dowden, Hutchinson and Ross, Stroudsburg, Pa. 1976), in the Introduction as follows:
The oxidation of organic and biological substrates under the influence of light, oxygen, and a sensitizer has been under investigation since the report by Fritzsche in 1867 that photoxy-genation of naphthacene yields a peroxide. Two general types of photosensitized oxygenation are observed: (1) the excited sensitizer serves to initiate a free-radical propagated autoxidation, and (2) the reactive intermediate is an electronically excited state of molecular oxygen (singlet oxygen) produced by the transfer of energy from the excited sensitizer to oxygen. An example of photochemically initiated autoxidation is the benzophenone-sensitized oxidation of isopropyl alcohol in the presence of oxygen, initially investigated by Baackstrom. Early photooxygenation reactions, which were sub-sequently shown to involve singlet oxygen, include the photo-oxygenation of rubrene investigated by Moureu, Dufraisse, and Dean . . . and the dye-sensitized photooxygenation of ergosterol investigated by Windaus and Brunken . . . . However, it was the classic synthesis of ascaridole from .alpha.-terpinene by Schenck and Ziegler in 1944 that prompted extensive preparative and mechanistic investigations of photooxygenation. PA1 Molecular oxygen, a ground-state triplet with paramagnetic and diradical-like properties, has two low-lying singlet excited states, .sup.1 .DELTA..sub.g and .sup.1 .SIGMA..sub.g.sup.30. As the transition of .sup.1 .DELTA..sub.g to .sup.3 .SIGMA..sub.g is spin-for bidden, .sup.1 .DELTA..sub.g is a relatively long-lived species. The .sup.1 .SIGMA..sub.g state is relatively short-lived with a spin-allowed transition to .sup.1 .DELTA..sub.g. The lifetime of the .sup.1 .SIGMA..sub.g.sup.+ is sufficiently short that all singlet oxygen chemistry in solution involves the .sup.1 .DELTA..sub.g.sup.+ state. PA1 In addition to photosensitization, several alternative methods for the generation of singlet oxygen have been developed: the reaction of sodium hypochlorite with hydrogen peroxide, the thermal decomposition of phosphite ozonides, the decomposition of epidioxides, the reaction of potassium superoxide in water, the self-reaction of sec-peroxy radicals, and microwave discharge through gaseous oxygen. PA1 The reactions of singlet oxygen with a wide variety of organic substrates are discussed. . . . Singlet oxygen exhibits three modes of reaction with alkenes: 1,4-cycloaddition with conjugated dienes to yield cyclic peroxides, an "ene"-type reaction to form allylic hydroperoxides, and 1,2-cycloaddition with olefins to give 1,2-dioxetanes, which cleave thermally to carbonyl-containing products. Other reactions of singlet oxygen include oxidation of sulfides to sulfox-deas and sulfones and addition to heterocycles such as pyrroles, furans, oxazoles, imidazoles, and thiophenes. Singlet oxygen also reacts with such biologically important substrates as fatty acids, purines, pyrimidines, DNA, PNA, amino acids (tyrosine, tryptophan, methionine, cystine, histidine) and various proteins. The possible role of singlet oxygen in biological oxidations has been considered by several investigators. PA1 "The acceptor has to be a compound that is polarly adsorbed by silica gel and which, under the given conditions, is not oxidized by normal oxygen but by activated oxygen. These requirements are met by leuco compounds of triphenylmethane dyestuffs. They possess the special advantage that, when oxidized, they are converted into intensely colored dyestuffs; therefore, they can serve as visible indicators for very small amounts of activated oxygen. The first experiments were carried out with .rho.-leucaniline, and the later experiments with leucomalachite green, which is more suitable because the blue color of malachite green formed during the oxidation of this compound is easier to distinguish from the above-mentioned rust-red coloration of the trypaflavine adsorbate arising upon extended irradiation." PA1 (a) upon subsequent reaction with molecular oxygen produces singlet molecular oxygen, or PA1 (b) upon subsequent reaction with a leucodye evokes a color change, or PA1 (c) upon subsequent reaction with a leucodye, whereby the sensitizer will assume a reduced form from which it can be returned to its original state by reaction with singlet oxygen resulting in the production of hydrogen peroxide. The hydrogen peroxide can be used as a signal to determine the amount of analyte or be used in chemical reactions that will signal the amount of analyte. PA1 (i) to form a dioxetan that decays upon heating to emit a detectable photon, or PA1 (ii) to form a peroxide that can either
In 1931, Kautsky and de Bruijn . . . proposed that dye-sensitized photooxygenation involved the transfer of electronic excitation energy from the excited sensitizer to oxygen to produce a "reactive, metastable state of the oxygen molecule." At the time of Kautsky's proposal, only the .sup.1 .SIGMA..sub.g.sup.+ excited state of oxygen had been observed spectroscopically, and this was assumed to be the reactive oxygen species. Following the report by Ellis and Kneser in 1933 of the .sup.1 .DELTA..sub.g state of oxygen, both states of oxygen were considered as possible reactants in photooxygenation. . . . Kautsky supported his proposed mechanism with a series of elegant experiments that should have put to rest the sensitizer-oxygen complex mechanism. It was observed that photooxygenation was possible even when the sensitizer and the acceptor were physically separated on different grains of silica gel, which demonstrated that only a diffusible molecule such as .sup.1 0.sub.2 could be the reactive species. In spite of these results, the Kautsky mechanism was almost totally disregarded and was not revived until the independent generation of singlet oxygen with NaOCl and H.sub.2 0.sub.2 and with the electrodeless discharge . . .
______________________________________ Electronic states of molecular oxygen Energy above Radiative life- ground State Lifetime in solu- time at zero pres- State (k cal) tion(s) sure ______________________________________ .sup.1 .SIGMA..sup.+.sub.g 37.5 10.sup.-9 to 10.sup.-12 7.1 s .sup.1 .DELTA..sub.g 22.5 10.sup.-3 to 10.sup.-6 45 min. .sup.3 .SIGMA..sup.-.sub.g 0 ______________________________________
Chapter 8 of the Schaap, supra, text, is a translation of an article by Kautsky et al., entitled: "Photosensitized Oxidation Mediated By A Reactive, Metastable State Of The Oxygen Molecule," in which singlet oxygen generated by the reaction of ground state oxygen and an excited photosensitizer, is transmitted to an acceptor. The acceptor was defined as follows (p. 35):
This is illustrated in the following: ##STR1##
Luminescence is a generic term covering a wide range of processes which produce light following electronic excitation through the absorption of any form of energy. Chemiluminescene or "cold light", may be defined as the emission of light as a result of an exergonic chemical reaction at temperatures below that required for incandescence. Bioluminescene or "living light" is a special case of chemiluminescence in biological systems in which the ongoing chemical reaction is catalysed by an enzyme or produced by photoproteins
The overall efficiency of light emission or quantum yield (.phi.) of a chemiluminescent reaction is defined, in Einsteins, as the number of photons produced by a mole of substrate. It is the product of the chemical .phi..sub.c, excitation .phi..sub.e, and fluorescence .phi..sub.f efficiencies as expressed below. EQU .phi.=.phi..sub.c .times..phi..sub.e .times..phi..sub.f
The quantum yield varies considerably from 0.88 for firefly bioluminescence to as low as 10.sup.-15. Typical values suitable for analytical applications are in the range 0.01 to 0.34.
There is much literature on tagging of a specific binding material with a compound that evokes a detectable signal. The signal may come from the decay of the label such as by emission of a radiolabeled form or by the decomposition of the label as in the case of luminescent labels. Other systems utilize biological processes, such as an enzyme-catalyzed reaction. The capabilities of such labeling systems are illustrated in Table A. Among the most sensitive of such systems are chemiluminescent immunoassays employing select classes of acridinium esters.
TABLE A ______________________________________ Detection Limits Of A Number Of Widely Used Labels In Immunoassay Typical Detection Immunoassay Label Limit/Mole ______________________________________ Radioisotopes .sup.3 H 1 .times. 10.sup.-16 .sup.125 I 1 .times. 10.sup.-18 Chemiluminescence Isoluminol 5 .times. 10.sup.-10 Acridinium Esters 2 .times. 10.sup.-18 Bioluminescence Firefly Luciferin-Lucif- 10.sup.-14 - erase 10.sup.-15 Enzyme with CL Detec- Peroxidase 8 .times. 10.sup.-17 tion Luminol/Enhancer Glucose-6-phosphate 1 .times. 10.sup.-18 dehydrogenase Isolu- minol Fluorescence Europium 2 .times. 10.sup.-17 (delayed fluorescence) Fluorescein 1 .times. 10.sup.-13 Enzyme .beta.-Galactosidase 2 .times. 10.sup.-18 Horseradish Peroxidase 3 .times. 10.sup.-18 Alkaline Phosphatase 5 .times. 10.sup.-19 ______________________________________
There is substantial literature about such labels, see e.g., McCapra, "Chemiluminescence of Organic Compounds," in Progress in Organic Chemistry, vol. 8, Carruthers and Sutherland ed., Wiley & Sons (1973); Kohen, Bayer, Wilechek, Barnard, Kirn, Colleins, Beheshti, Richardso and McCapra, "Development Of Luminescence-Based Immunoassays For Haptens And For Peptide Hormones," pp. 149-158, in Analytical Applications Of Bioluminescence and Chemiluminescence, Academic Press, Inc. (1984); Richardson, Kim, Barnard, Collins and McCapra, Clinical Chemistry, vol. 31, no. 10, pp. 1664-1668(1985); McCapra, "The Application of Chemilumio nescence in Diagnostics," 40.sup.th Conference of the American Association of Clinical Chemists, New Orleans, La, Jul. 28, 1988; McCapra, "The Chemiluminescence Of Organic Compounds," Quarterly Reviews, vol. 20, pp. 485-510 (1966); McCapra, "The Chemiluminescence Of Organic Compounds," Pure and Applied Chemistry, vol. 24, pp. 611-629(1970); McCapra, "The chemistry of bioluminescence," Proceedings Of Royal Society, vol. B215, pp. 247-278(1982); McCapra and Beheshti, "Selected Chemical Reactions That Produce Light," Bioluminescence and Chemiluminescence: Instruments and Applications, CRC Press, vol. 1, Chapter 2, pp. 9-37(1985); McCapra, "Chemiluminescent Reactions of Acridines," Chapt. IX, Acridines, R. M. Acheson, Ed., pp. 615-630, John Wiley & Sons, Inc. (1973); McCapra, "Chemical Mechanisms in Bioluminescence," Accounts Of Chemical Research, vol. 9, no. 6, pp. 201-208(Jun. 1976); and in many other publications and presentations on the subject.
The use of certain dye as labels is well recognized in the literature, see, e.g., Rinderknecht, Nature, 193, 4811, p. 167 (1962) who describes the labeling of proteins with fluoresceinisothiocyanate and dimethylaminonaphthalene sulphonyl chloride; Mann et al., Method of Enzymology, 26, pp. 28-42, see pp. 36-39 (1972) who describe fluorescein, .sup.14 C-labeling of amino groups, incorporation of dinitrophenyl and trinitrophenyl; and Riggs et al., American Journal of Pathology, 34, 6, pp. 1081-1097(1958) labeled serum with ##STR2## and used the two to determine by staining the presence of the antigen; Cherry et al., Stain Technology, "Evaluation Of Commercial Fluorescein Isothiocyanates Used In Fluorescent Antibody Studies," 44, 4, pp. 179-186 (1969) describe comparable information.
Molecular Probes, Inc., P.O. Box 22010, (4849 Pitchford Avenue) Eugene, Oreg. 97402-0414, offers an extensive variety of fluorescent probes for use in labeling applications. A substantial number of the labeling compound listed below are taken from their Handbook Of Fluorescent Probes and Research Chemicals.